U-rich elements drive pervasive cryptic splicing in 3' UTR massively parallel reporter assays

Area of Science:

• Molecular Biology
• Genetics
• RNA Biology

Background:

• Untranslated RNA sequences are crucial for gene expression regulation.
• Mechanisms of post-transcriptional regulation are not fully understood.
• AU-rich elements in 3' UTRs were previously shown to modulate mRNA expression.

Purpose of the Study:

• To investigate the mechanisms behind context-dependent regulation by AU-rich elements.
• To identify the source of variable mRNA expression observed in reporter assays.
• To understand the role of U-rich sequences in splicing and gene regulation.

Main Methods:

• Revisiting data from a prior massively parallel reporter assay (MPRA).
• Analyzing cryptic splicing events using reporter constructs.
• Investigating the influence of U-rich sequences on splice site selection and efficiency.

Main Results:

• Widespread cryptic splicing was discovered, originating from an unannotated splice donor in GFP to various acceptor sites in 3' UTRs.
• U-rich sequences were identified as potent, position-dependent regulators of splicing.
• Cryptic splicing impacts reporter expression, causing both increases and decreases, and affects other 3' UTR MPRA studies.

Conclusions:

• U-rich sequences are principal drivers of cryptic splicing.
• Cryptic splicing is a significant artifact in reporter assays, influencing gene expression measurements.
• Strategies to minimize cryptic splicing artifacts in reporter assays are provided.